Abstract: For more than 550,000 patients annually diagnosed with head and neck cancers worldwide, severe loss of salivary gland function (xerostomia) is an unavoidable outcome of radiation therapy. There are presently no reliable and safe pharmacologic treatments for the resolution or prevention of radiation-induced xerostomia. Efforts to study radiosensitivity to discover effective radioprotective and regenerative strategies have been hampered by the inability to culture salivary gland mimetics in vitro, due to loss of secretory acinar cell phenotype. The principal milestone of this proposal is to engineer functional human salivary gland tissue chips to overcome this obstacle. Our labs have pioneered the use of hydrogel encapsulation to culture salivary gland cells in vitro. We have successfully demonstrated salivary gland cell survival up to 1 month post- encapsulation. Furthermore, cells organize into structures with apicobasal polarity and express secretory acinar markers, including Mist1. Although these data are promising, secretory marker expression is reduced compared to the native gland. Furthermore, the macroscale nature of hydrogels precludes their high- throughput use. Thus, we will utilize our microbubble (MB) array technology as a high-throughput, modular platform for the tissue chips. MBs are micron-scale spherical cavities molded in polydimethylsiloxane (PDMS). MBs have the advantage of length scales and curvatures similar to the secretory acinar unit of glands, providing a niche that promotes cell-cell contact and the concentration autocrine and paracrine factors that have been shown to enhance tissue assembly. Furthermore, MBs can be integrated with other microphysiological systems such as endothelial, nerve, and immune system chips. During the UG3 phase of this project, the go/no-go criteria will be the use of the MB platform to develop human gland tissue mimetics capable of long-term secretory function. Specifically for UG3, Aim 1 will use genetically labeled mouse acinar and duct cells to identify culture characteristics that maximize gland tissue mimetic function. Acinar and duct cell seeding ratios and densities will be varied in ?blank?, extracellular matrix protein-functionalized, and in hydrogels all within MBs. Aim 2 (UG3) will validate the ability of human salivary gland cells to cellularly organize and maintain function in our previously developed macrogels and in within hydrogels in MBs, similarly to mouse cells in Aim 1. Our goal is to demonstrate functional human gland mimetic development in MB arrays by end of UG3. If successful, the UH3 phase will investigate hydrogel microenvironmental cues to further promote gland mimetic organization and function. Finally, Aim 3 will demonstrate the utility of gland mimetics by screening FDA-approved drugs to identify effective radioprotective agents. These compounds will be retroductally injected into mice to validate radioprotective potential. Successful development of salivary tissue chips will be transformative; by enabling in vitro analysis of functional gland mimetics, our ability to pursue therapeutic strategies, radioprotective and regenerative, will be dramatically improved.!